Abstract: Cancer cells survival depends on their ability to block cell death mechanisms. To evade apoptosis cancer cells block the intrinsic pathway of apoptosis by modulating the control and function of the BCL-2 protein family. The intrinsic or mitochondrial pathway of apoptosis is regulated by the BCL-2 family of proteins that include: 1) the pro-apoptotic multi-domain proteins BAX and BAK, 2) the anti-apoptotic proteins e.g. BCL-2, MCL-1, BCL- XL, and 3) the pro-apoptotic BH3 only proteins. Cancer cells commonly block apoptosis by upregulating the BCL- 2 anti-apoptotic proteins and/or downregulating or inhibiting the BCL-2 pro-apoptotic proteins. Thus, the BCL-2 family members have a critical role in regulating apoptosis in cancer and treatment response to chemotherapy. Targeting proteins of the intrinsic apoptosis pathway has been a highly attractive approach for the development of novel-anticancer therapies. Efforts have focused on the development of small molecules that inhibit selective BCL-2 anti-apoptotic proteins, induce release of BH3-only activator proteins and promote BAX/BAK-mediated apoptosis. This strategy has led to the recent FDA approval of the BCL-2 inhibitor Venetoclax and other molecules in clinical development. Our laboratory recently developed a direct activator of pro-apoptotic BAX, BTSA1 that induces apoptosis independently of BH3-only proteins. To broadly extend pro-apoptotic activity in cancer cells, we hypothesized to combine the BAX activator with selective anti-apoptotic proteins inhibitors. BAX has high affinity for the anti-apoptotic protein BCL-XL, a common oncogenic driver overexpressed in solid tumors, therefore we rationalized that the synthetic inhibition of BCL-XL anti-apoptotic protein will sensitize cancer cells to a co-treatment with our direct BAX activator BTSA1. To inhibit BCL-XL we will use Navitoclax a selective BCL-2/BCL-XL inhibitor, which is currently in clinical trials. Preliminary data indicates that the BTSA1/Navitoclax combination has a significant synergistic effect in solid tumor cell lines, as shown by the decrease in cell viability upon dual treatment. The goal for the proposed studies is to investigate the therapeutic potential and mechanism of rationally targeting both BAX and BCL-XL proteins in resistant tumors. In summary, this proposal will use an innovative approach to highlight how rationally co-targeting two anti-apoptotic survival mechanisms could broadly overcome resistance to apoptosis. We propose the following specific aims to achieve our goal: 1) evaluate the efficacy of co-targeting BAX and BCL-XL in human cancer cell lines, 2) determine the mechanism of action of co-targeting BAX and BCL-XL with BTSA1 and Navitoclax, and 3) evaluate and optimize the therapeutic potential and safety of co-targeting BAX and BCL-XL. With the help and expertise of my mentor and collaborators, I will be able to accomplish these goals and provide the first proof of concept of how rationally targeting two anti-apoptotic survival mechanisms can lead to enhance cell death in tumors, something that could be translated to more effective therapies for cancer patients.